WO2005082936A1 - Means for a quantitative detection of cytochrome c - Google Patents
Means for a quantitative detection of cytochrome c Download PDFInfo
- Publication number
- WO2005082936A1 WO2005082936A1 PCT/EP2005/002577 EP2005002577W WO2005082936A1 WO 2005082936 A1 WO2005082936 A1 WO 2005082936A1 EP 2005002577 W EP2005002577 W EP 2005002577W WO 2005082936 A1 WO2005082936 A1 WO 2005082936A1
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- WO
- WIPO (PCT)
- Prior art keywords
- cytochrome
- factor
- kit
- nadh
- reducing agent
- Prior art date
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
Definitions
- the invention relates to means for detecting cytochrome c release from mitochondria in a biological sample to be tested.
- Mitochondria play a central role in the regulation of cellular apoptosis through release of proteins into cytosol.
- Bcl-2 Since the anti-apoptotic protein Bcl-2 was found to reside in mitochondria, scientists started to consider this organelle as important player in apoptosis. Two main findings reinforced the connection between Bcl-2, mitochondria and apoptosis: Bcl- 2 was found to prevent the drop in mitochondrial membrane potential ( ⁇ m) observed during TNF-induced apoptosis in many cell types. In the meantime, Newmeyer et al . discovered that a mitochondrial factor was required for the activation of caspases.
- cytochrome c This factor, released from mitochondria during apoptosis, was later identified as cytochrome c.
- Two main apoptotic pathways have been described over the past few , years : the death-receptor pathway and the mitochondrial pathway. The first is engaged by death receptors which, upon binding to their appropriate ligands, form a death-inducing signalling complex, resulting in pro-caspase-8 activation.
- type I cells T-Lymphocytes
- caspase-8 cleaves downstream caspases that execute the cell.
- type II T-Lymphocytes
- Hepatocytes caspase-8 cleaves Bid, a BH3-only protein whose
- C-terminal fragment translocates to mitochondria to engage the mitochondrial pathway.
- the m ⁇ tochondrial pathway can also be activated in response to a large number of death stimuli including DNA damage, topoisomerase inhibition or trophic-factor depletion. This process culminates in the release of mitochondrial proteins from the intermembrane space into the cytosol.
- cytochrome c detection has a great interest for discovering pro- or anti-apoptotic drugs, integrated in screening strategies, alone or combined with other measurements ( ⁇ m, mitochondrial swelling, etc) .
- This type of investigations is commonly conducted using immunodetection (for instance Western blotting or ELISA.) as method of detection.
- Western blotting is a time consumming procedure (approximately 2 days) and a semi- quantitative method with poor accuracy.
- this method cannot be used for detailled analysis of the amount and kinetics of cytochrome c release under differing conditions, and cannot be used ror drug screening.
- ELISA quantification requires plate coating, use of pre-set formats and is not really user-friendly because of the multiple washing stages.
- HPLC has also been used for the quantitation of cytochrome c. Compared to Western blotting, the HPLC method is able to provide quantitative data. However samples have to be quantified sequentially and each quantification requires 20 minutes at least. ⁇ The inventors have found that by using cytochrome c- specific agents it was possible to determine cytochrome c concentration in different sub-cellular fractions by an enzymatic method. The aim of the invention is first to provide a tool to accurately quantify cytochrome c concentration in biological samples. It is another object of the invention to provide a ready-made kit, for performing the described assay method.
- the method requires: - adding to the studied sample an efficient amount of two redox couples for a cycling oxydo-reduction of cytochrome c, said couples consisting in an oxidizing agent, cytochrome c oxidase enzyme (COX) , and a reducing agent specific for cytochrome c with a co-factor, - measuring the oxidation of the co-factor which is oxidized during the redox cycle, the amount of the oxidative form thereof being correlated to the concentration of cytochrome c in the sample.
- COX cytochrome c oxidase enzyme
- COX cytochrome c oxidase enzyme
- the reducing agent specifically reduces cytochrome c and this reduction is detected by monitoring the concomitant oxidation of said co-factor.
- the oxidizing agent cytochrome c oxidase
- cytochrome c oxidase is added to the reaction medium to re-oxidize cytochrome c, allowing this cytochrome c to be used again by the reducing agent.
- the cycling redox system which is generated is strictly dependent on the presence of cytochrome c. Accordingly the amplification of the co-factor oxidation indicates that cytochrome c is present in the tested sample.
- the oxidation of the co-factor is handily measured by a biophysical system depending on the co-factor and allowing to distinguish the oxidized form from the reduced form (for example but not limited to, absorbance measurement ⁇ by molecular absorption spectrophotometry at 340 nm for NADH or NADPH detection) .
- the reducing agent is NADH-cytochrome c reductase or NADPH-cytochrome c reductase and the co-factor is NADH or NADPH respectively.
- said measurement is compared to measurements of known concentrations of standard cytochrome c.
- Said oxidizing and reducing agents and co-factors are, for example but not limited to, under liquid, dried or lyophilised form and obtained by purification of recombinant or natural compounds or by chemical synthesis.
- the detection may be performed on any biological sample suspected to contain cytochrome c, such as cellular extracts or organelles purified from primary cells, cell lines, tissues,, blood, organs or tumors that may or may not have been submitted to stress, particularly apoptosis-inducing stress.
- the detection is advantageously performed in supernatants obtained upon sedimenting mitochondria following incubation under various experimental conditions; or in cellular extracts obtained upon cytosol purification after cells incubation under various experimental conditions .
- the invention also relates to a kit for detecting cytochrome c in sample to be tested.
- a kit comprises: two redox couples for a cycling oxido-reduction of cytochrome c, said couples consisting in an oxidizing agent, i.e. cytochrome c oxidase enzyme, and a reducing agent, specific for cytochrome c, using a reduced co- factor.
- the reducing agent is advantageously a NADH-cytochrome c reductase or NADPH-cytochrome c reductase and the co-factor is NADH or NADPH respectively.
- said agents are, for example but not limited to, under liquid, dried or lyophilised form and obtained by purification of recombinant or natural compounds or by chemical synthetis.
- said kit further comprises a buffer.
- Said kit also comprises standard cytochrome c as a reference. • Said means are advantageous substitutes to immuno-assay, HPLC detection or Western blotting in order to detect cytochrome c in biological samples.
- the present invention is further illustrated by the 5 following examples and figures, which respectively represent: Fig. 1: Cycling enzyme assay for the detection of cytochrome c; Fig.
- NAD(P)H oxidation is dependent on the simultaneous presence of the various components of the 10 enzymatic cycle and is fully bloc :ed upon cytochrome c oxidase inhibition;
- Fig. 3 At constant cytochrome c concentration, the rate of the cycling reaction is dependent on the amount of added enzymes; 15 -
- Fig. 4 At saturating enzyme concentrations, the rate of the cycling assay only depends on cytochrome c concentration;
- Fig. 5 Saturating enzyme concentration permits to detect low concentration of cytochrome c in mitochondrial 20 supernatants .
- measurements are performed in transparent flat-bottom 96-well microplates in a final volume of 220 microliter.
- Stock solution of cytochrome c and NADH or NADPH are prepared in distilled water.
- Enzyme A is a cytochrome c reductase that catalyses cytochrome c reduction and concomitant co-factor (NADH or NADPH) oxidation. Oxidized cytochrome c is thus reduced by the reductase.
- Enzyme B is the cytochrome c oxidase: it oxidizes reduced cytochrome c and transfers electron to molecular oxygen. The presence of both enzymes allows for a redox cycle using cytochrome c. The rate of cycling becomes limited by cytochrome c in the presence of an excess of both enzymes.
- NAD(P)H oxidation is then directly proportionnal to the amount of available cytochrome c.
- Cyanide (KCN) is a cytochrome c oxidase inhibitor. NAD(P)H oxidation is dependent on the simultaneous presence of the various components of the enzymatic cycle and is fully blocked upon cytochrome c oxidase inhibition. Measurements are performed by absorption spectrophotometry at 340 nm (fig.2) . Complete medium : 300 ⁇ M NADH, 300 ⁇ U NADH-cytochrome c reductase, 300 ⁇ U cytochrome c oxidase, 2 ⁇ M cytochrome c; 1. NADH only; 2.
- Measurements are performed by absorption spectrophotometry at 340 nm (fig.3) .
- the oxidation of NADH 300 ⁇ M correlates with enzyme concentration.
- ' NADH-cytochrome c reductase is in excess compared to cytochrome c oxidase, and the rate of the reaction is dependent on this latter enzyme. A large excess of both enzymes is therefore required to avoid any interference that may result from enzymes potentially added with studied samples .
- the rate of the cycling assay only depends on cytochrome c concentration. Measurements are performed by absorption spectrophotometry at 340 nm (Fig.4) .
- NADH 300 ⁇ M
- a linear relationship exists between the rate of NADH consumption and the cytochrome c concentration. It makes this reaction a simple and convenient method to quantify cytochrome c.
- Washed mitochondria were layered on top of two successive Percoll gradients, consisting of three layers of 18%, 30% and 60% (w/v) Percoll in medium B (0.3 M saccharose, 0.2 mM EGTA, 10 mM TE; pH 6.9). After centrifugation (8740 g, 10 ' min), the fraction containing intact mitochondria was collected from the 30%/60% interface, washed with medium A (8740 g, 10 min) and the pellet resuspended in 500 ⁇ L medium A. Protein concentration was determined by BCA assay.
- Purified mitochondria (4 mg protein/mL) were then incubated in medium C (0.2 M saccharose, 5 mM- succinate, 10 ⁇ M EGTA, 1 mM H 3 P0 4 , 2 ⁇ M Rotenone and 10 mM Tris-MOPS; pH 7.4) for 30 min at room temperature with 500 ⁇ M calcium chloride or 5 ⁇ g/mL alamethicin as- inducers of cytochrome c release.
- medium C 0.2 M saccharose, 5 mM- succinate, 10 ⁇ M EGTA, 1 mM H 3 P0 4 , 2 ⁇ M Rotenone and 10 mM Tris-MOPS; pH 7.4
- 500 ⁇ M calcium chloride or 5 ⁇ g/mL alamethicin as- inducers of cytochrome c release.
- Treated mitochondria were centrifuged (6800 g, 10 min at 4°C) and supernatants were about 20 fold-concentrated on 10,000 Da concentrator microtubes for
- Each sample (20 microliter) ⁇ was added to 200 microliter reaction solution (300 ⁇ M NADH in 180 ⁇ L assay buffer, 1 mU NADH- cytochrome c reductase in 10 ⁇ L enzyme buffer, 1 mU cytochrome c oxidase in 10 ⁇ L enzyme buffer); 1. Distilled water; 2.
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Abstract
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Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP05715949A EP1723176A1 (en) | 2004-02-26 | 2005-02-24 | Means for a quantitative detection of cytochrome c |
US10/590,651 US20070275432A1 (en) | 2004-02-26 | 2005-02-24 | Means for a Quantitative Detection of Cytochrome C |
JP2007500190A JP2007536914A (en) | 2004-02-26 | 2005-02-24 | Cytochrome c quantitative detection means |
Applications Claiming Priority (2)
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---|---|---|---|
US54756404P | 2004-02-26 | 2004-02-26 | |
US60/547,564 | 2004-02-26 |
Publications (1)
Publication Number | Publication Date |
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WO2005082936A1 true WO2005082936A1 (en) | 2005-09-09 |
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PCT/EP2005/002577 WO2005082936A1 (en) | 2004-02-26 | 2005-02-24 | Means for a quantitative detection of cytochrome c |
Country Status (4)
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US (1) | US20070275432A1 (en) |
EP (1) | EP1723176A1 (en) |
JP (1) | JP2007536914A (en) |
WO (1) | WO2005082936A1 (en) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
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EP2057264A1 (en) * | 2006-08-23 | 2009-05-13 | Tautheta Instruments LLC | Metabolic monitoring device |
US20120040387A1 (en) | 2009-01-19 | 2012-02-16 | Asahi Kasei Pharma Corporation | Method and reagent for measuring mevalonic acid, 3-hydroxymethylglutaryl coenzyme a, and coenzyme a |
Family Cites Families (1)
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JPS63116700A (en) * | 1986-11-01 | 1988-05-20 | Imuno Baion:Kk | Analytical method for trace substance |
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2005
- 2005-02-24 US US10/590,651 patent/US20070275432A1/en not_active Abandoned
- 2005-02-24 WO PCT/EP2005/002577 patent/WO2005082936A1/en active Application Filing
- 2005-02-24 JP JP2007500190A patent/JP2007536914A/en active Pending
- 2005-02-24 EP EP05715949A patent/EP1723176A1/en not_active Withdrawn
Non-Patent Citations (8)
Title |
---|
"A NEW QUANTITATIVE ASSAY FOR CYTOCHROME C RELEASE IN APOPTOTIC CELLS", CELL DEATH AND DIFFERENTIATION, EDWARD ARNOLD, OXFORD, GB, vol. 10, no. 7, July 2003 (2003-07-01), pages 853 - 855, XP001206771, ISSN: 1350-9047 * |
CATINO J J ET AL: "Microtiter assay useful for screening of cell-differentiation agents.", JOURNAL OF THE NATIONAL CANCER INSTITUTE. 17 AUG 1988, vol. 80, no. 12, 17 August 1988 (1988-08-17), pages 962 - 966, XP009048730, ISSN: 0027-8874 * |
CHRZANOWSKA-LIGHTOWLERS Z M ET AL: "A microtiter plate assay for cytochrome c oxidase in permeabilized whole cells.", ANALYTICAL BIOCHEMISTRY. OCT 1993, vol. 214, no. 1, October 1993 (1993-10-01), pages 45 - 49, XP002332122, ISSN: 0003-2697 * |
ISHIHARA T ET AL: "Detection of cytochrome C by means of surface plasmon resonance sensor", SENSORS AND ACTUATORS B, ELSEVIER SEQUOIA S.A., LAUSANNE, CH, vol. 91, no. 1-3, 1 June 2003 (2003-06-01), pages 262 - 265, XP004424428, ISSN: 0925-4005 * |
LU W ET AL: "Use of Prussian blue/conducting polymer modified electrodes for the detection of cytochrome C", ELECTROANALYSIS, vol. 10, no. 7, June 1998 (1998-06-01), pages 472 - 476, XP009048727, ISSN: 1040-0397 * |
LU WEN ET AL: "Detection of cytochrome c using a conducting polymer mediator containing electrode", ELECTROANALYSIS, vol. 8, no. 3, 1996, pages 248 - 252, XP009048726, ISSN: 1040-0397 * |
PICKLO M J ET AL: "High-pressure liquid chromatography quantitation of cytochrome c using 393 nm detection.", ANALYTICAL BIOCHEMISTRY. 15 DEC 1999, vol. 276, no. 2, 15 December 1999 (1999-12-15), pages 166 - 170, XP002332121, ISSN: 0003-2697 * |
SMITH L: "Spectrophotometric assay of cytochrome c oxidase.", METHODS OF BIOCHEMICAL ANALYSIS. 1955, vol. 2, 1955, pages 427 - 434, XP009048732, ISSN: 0076-6941 * |
Also Published As
Publication number | Publication date |
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EP1723176A1 (en) | 2006-11-22 |
JP2007536914A (en) | 2007-12-20 |
US20070275432A1 (en) | 2007-11-29 |
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